Mutagenicity and antimutagenicity of Salacia crassifolia (mart. Ex. Schult.) G. Don. evaluated by Ames test / Mutagenicitade e antimutagenicidade de Salacia crassifolia (mart. Ex. Schult.) G. Don. avaliadas pelo teste de Ames

Abstract Salacia crassifolia (Mart. Ex. Schult.) G. Don. is a bush which belongs to Celastraceae family and occurs specially in Brazilian Cerrado. Its leaves, stem, seeds and fruits are popularly used for several medicinal purposes, such as antitumoral, antirheumatic, anti-inflammatory and antimicrobial. In this study, the mutagenic and antimutagenic activities of S. crassifolia stem bark fractions (hexane, ethyl acetate and hydroalcoholic) were evaluated by the Ames mutagenicity assay in Salmonella typhimurium TA98 and TA100 strains. By the obtained results, all S. crassifolia fractions did not significantly increase the number of prototrophic revertants for histidine (His+) in both S. typhimurium strains tested (p > 0.05), suggesting absence of mutagenicity. Regarding antimutagenicity, the fractions ethyl acetate and hydroalcoholic significantly decreased the number of His+ revertants colonies induced by positive control for strain TA98 (p < 0.05), demonstrating protection against mutagenicity induced by 4-nitroquinolile1-oxide, whereas the hexane fraction did not show antimutagenic effect in this strain. In the TA100 strain, all fractions of S. crassifolia protected DNA against the harmful action of sodium azide, and the hexane fraction exhibited the greatest protection in this work. Thus, it's possible conclude that the fractions of S. crassifolia tested in this study could be used in chemoprevention.

Resumo Salacia crassifolia (Mart. Ex. Schult.) G. Don. é uma árvore que pertence à família Celastraceae e ocorre especialmente no Cerrado Brasileiro. Suas folhas, caule, sementes e frutos são popularmente utilizados para vários fins medicinais, tais como antitumoral, antirreumático, anti-inflamatório e antimicrobiano. Neste estudo, nós avaliamos as atividades mutagênica e antimutagênica de frações da casca do caule de S. crassifolia (hexânica, acetato de etila e hidroalcoólica) pelo ensaio de mutagenicidade de Ames em Salmonella typhimurium, cepas TA98 e TA100. Pelos resultados obtidos todas as frações de S. crassifolia não aumentaram significativamente o número de revertentes prototróficas para histidina (His+) em ambas as cepas de S. typhimurium testadas (p > 0.05), sugerindo ausência de mutagenicidade. Em relação à antimutagenicidade, as frações acetate de etila e hidroalcoólica reduziram significativamente o número de colônias revertentes His+ induzidas pelo controle positive para a cepa TA98 (p < 0.05), demonstrando sua ação protetora contra a mutagenicidade induzida por 4-nitroquinolile1-oxide, enquanto a fração hexânica não demonstrou efeito antimutagênico nesta cepa. Na cepa TA100, todas as frações de S. crassifolia protegeram o DNA contra a ação lesiva de azida sódica, e a fração hexânica exibiu a maior proteção desse trabalho. Assim, concluímos que as frações de S. crassifolia testadas neste estudo poderiam ser utilizadas em quimioprevenção.

Study on lingual mucosa carcinogenesis of C57BL/6 mice induced by 4-nitroquinoline 1-oxide / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed to induce carcinogenesis of lingual mucosa in C57BL/6 mice by feeding them 4-nitroquinoline 1-oxide (4NQO) solution.</p><p><b>METHODS</b>A total of 85 C57BL/6 mice were randomly divided into distilled water control group (DD group, n=5), 1,2-propylene glycol control group (PG group, n=5), and experimental group (EP group, n= 75). The mice in the experimental group were medially fed in 15 cages. By contrast, the mice in DD, EP, and PG groups were watered with distilled water, 50 mg.L-1 4NQO solution, and 1,2-propylene glycol solution. The mice in EP group were executed every two weeks from week 0, and the mice in the control groups were sacrificed at the 28th week. The mice were weighed. Mucosal lesions were measured by macroscopic observation and histopathologic detection.</p><p><b>RESULTS</b>One mouse in EP group died of unknown reason. The weight of the mice in EP group presented weight loss compared with the mice in DD and PG groups after the 24th week. Seventy-nine macroscopic lesions were observed in the lingual mucosa, oral floor, and upper palatal and buccal mucosa. A total of 70 macroscopic lesions (88.6%) were located in the lingual mucosa. Mucosal lesions changed from simple hyperplasia to squamous cell carcinomas. Well-differentiated squamous cell carcinomas were observed in all mice of EP group by pathological section at the 28th week. No lesion was found in the mice of DD and PG groups.</p><p><b>CONCLUSION</b>The animal model of lingual squamous cell carcinomas was successfully established. The periods from 12th to 16th week and 20th to 28th week were the ideal times for the research on pathogenesis of early and medial-advanced stage during carcinogenesis of squamous cell carcinomas.</p>

Myeloid-derived suppressor cell expression and significance in peripheral blood and tongue lesions of mouse / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To explore the myeloid-derived suppressor cell (MDSC) expression in the peripheral blood and lesions of 4NQO-induced tongue carcinoma in mouse.</p><p><b>METHODS</b>The established 4NQO mouse model was used to analyze the distribution of MDSC and T cell subsets in the peripheral blood by flow cytometry. The relations of MDSC with T cell subsets and CD4⁺/CD8⁺ changes were evaluated. The distribution of MDSC in the lesions of tongues was analyzed by immu- nohistochemistry, and the expression of arginase 1 (ARG-1) in tongue tissues was detected by real-time polymerase chain reaction.</p><p><b>RESULTS</b>During tumor progression, a significant increase was observed in the frequency of MDSC in the peripheral blood of 4NQO treated mice (P < 0.01). The frequency of MDSC was positively correlated with systemic CD3⁺CD8+T cells but negatively correlated with the CD4⁺/CD8⁺ ratio. Squamous cell carcinomas were extensively infiltrated with MDSC, whereas dysplastic area and normal tongue mucosa had only sparse MDSC infiltration. The majority of MDSCs were located in the stroma, particularly along the tumor invasive front. Moreover, 4NQO-treated mice showed significantly higher ARG-1 mRNA levels in the tumor site (P<0.01).</p><p><b>CONCLUSION</b>MDSC may contribute to oral tumor progression and represents a potential target for immunotherapy of oral cancer.</p>

Preventive effects of tomato pulp on oral pre-neoplastic changes induced by 4-Nitroquinoline-1-oxid in the rat

Squamous cell carcinoma [SCC] is the most frequent oral cancer. Protective effects of the consumption of vegetables and fruits on various forms of cancer including oral cancer have been determined. Tomato [Solanum lycopersicum L.] because of its lycopene and bioflavonoids contents possesses anti-carcinogenic properties. The aim of this study was to evaluate the preventive effects of tomato pulp on pre-neoplastic changes induced by 4-Nitroquinoline-1-oxid [4-NQO] in epithelial cells of lingual mucosa in the rats. Forty-eight male Wistar rats were randomly allocated into four equal groups. Group 1 served as control. Groups 2 to 4 assigned to receive 30 ppm 4-NQO in drinking water for 12 consecutive weeks. When the feeding of 4-NQO was started to the rats of groups 3 and 4, they received tomato pulp [20 and 40 ml/kg bw] daily through the oral gavage. Finally, histological evaluations for carcinogenesis were performed for tongues epithelial tissue. There were no pathological alterations in epithelial tissue of lingual mucosa in control rats. In the epithelial cells of lingual mucosa of 4-NQO treated rats, premalignant alterations appeared after 12 weeks of the last application of the drug. Administration of tomato pulp at both doses [20 and 40 ml/kg bw] during the experiment reduced the severity of the lesions, as well as caused a significant reduction in the frequency of pre-neoplastic lesions of tongue epithelial cells [P= 0.024 and P= 0.008]. The incidence of severe epithelial cells dysplasia of lingual mucosa in the high dose treatment group was significantly smaller than of low dose treatment group [P= 0.037]. The results obtained showed that tomato pulp is effective in inhibiting the development of neoplasms in epithelial cells of lingual mucosa induced by 4-NQO in the rat

Evaluation of chemoprevention effect of systemic celecoxib on induction of tongue neoplasms in rat

Cyclo-oxygenase-2 [COX-2] specific inhibitors were examined for predication or treatment of different tumors and it is indicated that COX-2 specific inhibitors play an important regulatory role in apoptosis of tumoral tissues. Therefore, the present study was designed in order to examine the preventive effects of a COX-2 specific inhibitor called. celecoxib on 4-nitroquinoline 1-oxide [4NQO]-induced squamous cell carcinoma on rat. In this experimental study, 30 Sprague Dawley rats [with the age of 3- 3.5 months] were selected and divided into three groups. In order to induce lingual carcinoma, 4NQO powder was prepared 3 times a week for each cage. In this study, celecoxib power was mixed with a basic food [basal diet] in order to examine the systematic effect. Tongue samples were sent to laboratory for immunohistochemical [IHC] staining and histological examination. Based on morphological criteria and the ratio of apoptosis to cell proliferation, the prevalence of tongue precancerous lesions was reduced significantly by celecoxib. Celecoxib systematic has inhibitory effects on the 4-nitroquinoline 1-oxide [4NQO]-induced squamous cell carcinoma of tongue. The effect of celecoxib is probably via suppression of cell proliferation and induction of apoptosis

Expression of cytokeratin 19 and connexin 43 in 4-nitroquinoline-l-oxide-induced rat tongue carcinogenesis / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the expression of cytokeratin 19(CK19) and connexin 43(Cx43) in various stages of oral carcinogenesis and investigate the relationship of CK19 and Cx43 in the process of oral cancer.</p><p><b>METHODS</b>4-nitroquinoline-1-oxide(4NQO) was used to induce oral carcinogenesis in the mucosa of SD rats and immunohistoche-mical technique was used to study the expression of CK19 and Cx43 in various stages of oral carcinogenesis.</p><p><b>RESULTS</b>The CK19 positive staining were distributed in the basal cell layer in the normal rat lingual mucosa. While CK19 positive staining were distributed in cytoplasm of supra-basal layers in the mild dysplasia, moderate dysplasia and severe dysplasia. In oral squamous cell carcinoma(OSCC) tissue, CK19 were expressed in all the stratum of epithelium. The positive rate of CK19 in normal, mild, moderate, severe dysplasia and OSCC tissues were respectively 30.00%, 50.00%, 58.33%, 80.00%, and 91.67%. With the lesions getting worse, the positive rate and the intensity of CK19 raised significantly (P<0.05). In normal tongue mucosa, Cx43 proteins were mainly expressed in the membrane of the epithelial cells of the rat tongue. It was weakly positive in the basal cell layer, increased in the stratum spinosum and stratum granulosum, and negative in the stratum corneum. Compared with normal epithelia, the expression of Cx43 in dysplastic and OSCC epithelia decreased significantly. The positive rate of Cx43 in normal, mild, moderate, severe dysplasia and OSCC tissues were respectively 100.00%, 85.71%, 66.67%, 40.00%, and 33.33%. The expression of Cx43 was significantly decreased with severity increasing (P<0.05).</p><p><b>CONCLUSION</b>The expression of CK19 protein significantly increases with the development of rat tongue carcinoma, suggesting that CK19 is associated with carcinogenesis. The expression of Cx43 protein dramatically decrease with the development of rat tongue carcinoma, suggesting that the abnormal expression of Cx43 protein is associated with oral mucosa carcinoma origination. The expression of CK19 and Cx43 has negative correlation. Combined detection of CK19 and Cx43 has an important role in the early diagnosis of OSCC and can help to improve the sensitivity and specificity of the early diagnosis of OSCC.</p>

Expression of connexin 43 in tongue carcinogenesis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the expression of connexin 43 (Cx43) in various stages of oral carcinogenesis and explore the relation between Cx43 and oral mucous carcinogenesis.</p><p><b>METHODS</b>4-nitroquinoline-1-oxide (4NQO) was used for inducing oral carcinogenesis in SD rats. Tissue samples were obtained from various stages of the disease including normal oral mucosa, precancerous lesions and oral squamous cell carcinoma. Immunohistochemical method was used to determine the expression of Cx43 in various stages of oral carcinogenesis.</p><p><b>RESULTS</b>In the normal rat lingual mucosa, immunohistochemical staining of Cx43 protein was mainly found in the cell membrane, weakly positive in the basal cell layer, increased in stratum spinosum and stratum granulosum, but was negative in the stratum corneum of normal epithelia. Compared with normal epithelia, was significantly decreased in dysplastic and cancerous oral epithelia the staining. The positive rates of Cx43 were respectively 100.00% (10/10), 85.71% (12/14), 66.67% (8/12), 40.00% (4/10), and 33.33% (4/12) in tongue carcinogenesis (in normal, mild, moderate and severe dysplasia, and squamous cell carcinoma tissues). The differences were statistically significant (P<0.05).</p><p><b>CONCLUSION</b>Expression level of Cx43 protein was dramatically decreased with the development of rat tongue carcinoma induced by 4NQO, suggesting that abnormal expression of Cx43 protein is involved in oral mucosa carcinogenesis. Decreased Cx43 expression is an early sign of oral mucosa carcinogenesis.</p>

Expression of survivin, Bcl-2 and p53 during 4-nitro-quinoline 1-oxide-induced rat tongue carcinogenesis / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the expression of survivin during oral carcinogenesis and its relationship with the expression of Bcl-2 and p53.</p><p><b>METHODS</b>Two-step immunohistochemical method was employed to detect the expression of survivin, Bcl-2 and p53 in 60 rat tongue carcinogenesis specimens induced by 4-nitro-quinoline 1-oxide (4NQO).</p><p><b>RESULTS</b>Survivin was present in 1/36 cases of normal mucosa, 6/11 cases of oral epithelial dysplasia and 10/11 cases of oral carcinoma. There were significant difference in the expression of survivin among normal mucosa, oral epithelial dysplasia and carcinoma (P < 0.001). While in the 17 cases of positive expression of survivin, Bcl-2 was present in 12 cases and p53 was present in 8 cases. The expression of survivin in oral epithelial dysplasia and carcinoma were significantly higher than that in normal oral mucosa (P < 0.01). The expression of survivin was positively correlated to the expression of Bcl-2 and p53 in rat tongue (P < 0.01).</p><p><b>CONCLUSIONS</b>The expression of survivin may play an important role in oral carcinogenesis. Survivin, Bcl-2 and p53 may be synergetic in the carcinogenesis of oral cancer.</p>

Establishment and identification of biological characteristics of rat monoclone cell line Rca-B / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To establish a monoclone cell line of squamous cell carcinoma (SCC) in rat buccal mucosa and to study its biological characteristics.</p><p><b>METHODS</b>SCC in rat oral mucosa was induced by adding 4-nitroquinoline-1-oxide (4NQO) into the SD rats' drinking water, and the cancer cells were then cultured to obtain mixed cells in vitro. The mixed tumor cells were purified by mono cell cloning method. The biological characteristics of the cells were studied by microscope and electronic microscope observation, chromosome analysis, Methyl thiazolyl tetrazolium (MTT) test, flow cytometry assay and immunohistochemistry staining. Hypodermic inoculations of the cells in nude mice and injection of the cells by nude mice tail veins were performed to observe the tumor formation and long distance metastasis.</p><p><b>RESULTS</b>The morphology proved that the cell line was squamous cell carcinoma cells, which were cultured from one cell. The population doubling time for passage 65 cells was 25.44 hours. The cells in S-phase accounted for 20.13% of the cell cycle. The chromosome modal number was 84. All the cells expressed the proteins of cytokeratin and vimentin. The xenograft rate and the tumor metastatic rate to the lung were 100% in nu/nu BALB/C mice, but the homograft rate was zero in SD Rats.</p><p><b>CONCLUSIONS</b>Rca-B was a typical oral squamous cell carcinoma cell line derived from Sprague-Dawley rat buccal mucosa carcinoma, and the cell line has high metastatic potential and its biological characteristics were well ascertained.</p>

Influence of 1.8 GHz microwave on DNA damage induced by 4 chemical mutagens / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To observe the influence of 1.8 GHz microwave (MW) specific absorption rate (SAR, 3 W/kg) on human lymphocytes DNA damage induced by 4 chemical mutagens [mitomycin C (MMC), bleomycin (BLM), methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO)].</p><p><b>METHODS</b>Comet assay in vitro was used to detect human lymphocyte DNA damage induced by 1.8 GHz MW, 4 chemical mutagens, and MW plus 4 chemicals 0 h and 21 h respectively after exposure. The time exposed to MW or mutagens was 2 h or 3 h respectively. The results were showed by tail length (TL) and tail moment (TM).</p><p><b>RESULTS</b>The difference of DNA damage between MW group and control group was not statistically significant (P > 0.05). DNA damages in MW plus MMC groups and MW plus 4NQO groups were significantly greater than those in the corresponding concentrations of MMC groups and 4NQO groups (P < 0.01 or P < 0.05). However, MW did not enhance DNA damage induced by MMS and BLM (P > 0.05).</p><p><b>CONCLUSION</b>Exposure to 1.8 GHz (SAR, 3 W/kg) microwave may not induce human lymphocyte DNA damage, but could enhance DNA damage induced by MMC and 4NQO.</p>

Dietary protocatechuic acid during the progression phase exerts chemopreventive effects on chemically induced rat tongue carcinogenesis.

The modifying effects of dietary administration of protocatechuic acid (PCA) during the progression phase of tongue carcinogenesis initiated with 4-nitroquinoline 1-oxide (4-NQO) were investigated in male F344 rats. For tumor progression we developed a new animal model, where rats initiated by 4-week treatment of 20 ppm 4-NQO in drinking water, received four cycles of 20 ppm 4-NQO to induce advanced tongue cancer (one cycle: 2 weeks of 4-NQO followed by 2 weeks of tap water), starting at 14 weeks after the initiation. In this model, metastasis of tongue cancer occurred in lungs. Starting two weeks before the cycle treatment with 4-NQO, animals were fed the 2000 ppm PCA containing diet and continued on this diet until the end of the study. At the termination of the experiment (week 32), the incidences of tongue neoplasms and preneoplastic lesions, polyamine levels in the tongue tissue, and cell proliferation activity estimated by morphometric analysis of silver-stained nucleolar organizer regions protein were compared among the groups. Feeding with PCA containing diet during the progression phase significantly decreased the occurrence of advanced tongue squamous cell carcinoma with metastasis (P<0.05) and preneoplasia (hyperplasia and dysplasia) (P<0.001). In addition, PCA exposure decreased polyamine levels in the tongue tissue (P<0.001) during progression phase. Our results suggest that dietary PCA inhibits progression of 4-NQO-induced oral carcinogenesis, and such inhibition might be related to suppression of cell proliferation by PCA.